Fig 2: Knockdown of PTPN2 promotes IFN-γ-induced expression of p-STAT1, p-STAT3, and p-STAT5 along with MHC-I, MHC-II, and PD-L1 and potentiates anti-PD-L1 response. (A) Western blots show IFN-γ-induced p-STAT1, p-STAT3, and p-STAT5 expression in BRAF-mutant and BRAF-wildtype melanoma cell lines under conditions of nontargeting control siRNA (NTC) or specific PTPN2 siRNA (siPTPN2) transfection. Knockdown efficiency was confirmed by examining PTPN2 expression and actin was used as a loading control. (B) Western blots show IFN-γ-induced MHC-I, MHC-II, and PD-L1 expression in BRAF-mutant and BRAF-wildtype melanoma cell lines under conditions of nontargeting control siRNA (NTC) or specific PTPN2 siRNA (siPTPN2) transfection. (C) Schema for the in vivo experiment with results shown in panels D and E. (D) Tumor growth curves of YUMM1.1 with 5 mice in each group (mean ± SEM) with anti-PD-L1, or isotype control treatment under shPTPN2 conditions. (E) Waterfall plot of YUMM1.1 tumors with anti-PD-L1, or isotype control treatment under shPTPN2 conditions. The data are expressed as the percentage of tumor volume change from baseline.

Fig 3: Dephosphorylation of Flk-1 by TC-PTP increases the sensitivity to UVB-induced apoptosis in keratinocytes.a Seven potential tyrosine phosphorylation sites of Flk-1. b Western blot analysis of Flk-1 tyrosine phosphorylation at seven different sites in IPKs from both genotypes. Cells were collected at the indicated time after UVB irradiation (10 mJ/cm2). Total cell lysates were isolated and resolved by SDS-PAGE and immunoblotted with antibodies specific for pFlk-1 Y799, Y949, Y994, Y1025/1057, Y1173, or Y1212. c Western blot analysis of pFlk-1 (Y1173) in IPKs from both genotypes. Cells were collected at the indicated time after UVB irradiation (10 mJ/cm2). Total cell lysates were isolated and analyzed. d–e Western blot analysis of pFlk-1 (Y1173) in TC-PTP/KO IPKs in response to UVB irradiation. TC-PTP/KO IPKs were incubated with Flk-1 inhibitors, (d) SU5416 (2 µM) or (e) ZD6474 (2 µM) for 1 h before UVB irradiation (10 mJ/cm2). Cells were incubated with an inhibitor again for 1 h following UVB irradiation at which time the total cell fractions were extracted. f Effect of Flk-1 inhibition on cell viability after UVB exposure. TC-PTP/WT IPKs and TC-PTP/KO IPKs were cultured with SU5416 (2 µM) or ZD6474 (2 µM) for 1 h before UVB irradiation (10 mJ/cm2). Cell viability was analyzed by WST-1 assay 18 h after UVB irradiation. The results are the mean ± standard deviation from three independent experiments. *P < 0.05 by T test for equality of means. g–h TC-PTP/WT IPKs and TC-PTP/KO IPKs were cultured with SU5416 (2 µM) or ZD6474 (2 µM) for 1 h before UVB irradiation (10 mJ/cm2). Apoptotic cells were analyzed by TUNEL assay 18 h after UVB irradiation. g Representative TUNEL staining (red) from TC-PTP/WT IPKs and TC-PTP/KO IPKs after UVB exposure. Nuclei were stained with DAPI (blue). Scale bar, 20 µm. h Quantitative analysis of the percentage of TUNEL-positive cells from TC-PTP/WT IPKs and TC-PTP/KO IPKs. The results are the mean ± standard deviation from three independent experiments. *P < 0.05 by T test for equality of means. i Western blot analysis of cleaved PARP and cleaved caspase 3 in TC-PTP/WT IPKs and TC-PTP/KO IPKs. IPKs from both genotypes were cultured with SU5416 (2 µM) or ZD6474 (2 µM) for 1 h before UVB irradiation (10 mJ/cm2). Cells were collected at 12 h after UVB irradiation.

Fig 4: Effect of TC-PTP deletion on UVB-induced apoptosis.a–b Apoptotic response of the epidermis from K14Cre.Ptpn2w/w and K14Cre.Ptpn2fl/fl mice. Groups of mice (n = 3) were irradiated with UVB at 120 mJ/cm2 and sacrificed 24 and 48 h after irradiation. Skin sections from each mouse were collected and evaluated to measure apoptosis by immunostaining with caspase-3. a Representative phase-contrast image depicting active caspase-3 (dark spots) in the epidermis from both mouse genotypes before and after UVB irradiation. Scale bar: 100 μm. b Quantitative analysis of caspase-3-positive cells per centimeter of epidermis in both genotypes after UVB irradiation. *P < 0.05 by Mann–Whitney U test. c Western blot analysis of Bcl-xL and Bax in the epidermis from both genotypes following UVB irradiation at 120 mJ/cm2. Mice were sacrificed at the indicated time after UVB exposure and the total epidermal cell lysates were prepared.

Fig 5: Activation of Flk-1/JNK signaling axis in the epidermis of TC-PTP- deficient mice following UVB exposure.a Western blot analysis of pFlk-1 (Y1173) and pJNK in the epidermis from K14Cre.Ptpn2w/w and K14Cre.Ptpn2fl/fl mice following UVB irradiation at 120 mJ/cm2. Mice were sacrificed at the indicated time after UVB exposure and total epidermal cell lysates were prepared. b Schematic diagram of the regulation of Flk-1/JNK signaling by TC-PTP following UVB irradiation. UVB triggers the phosphorylation of Flk-1 (Y1173) which is a direct target of TC-PTP. In the absence of TC-PTP, Flk-1 phosphorylation on Y1173 can activate JNK signaling, resulting in the promotion of UVB-induced cell survival. TC-PTP contributes to increased epidermal apoptosis in response to UVB exposure by dephosphorylating Flk-1, which may serve as part of an initial protective mechanism against UVB radiation damage.